1. Field of the Invention
The present invention relates generally to a set of synthetic immunointeractive molecules referred to herein as “demibodies” which are useful in targeting particular cells in a subject. More particularly, the present invention provides a set of demibodies wherein at least two molecules from within the set, each specific for a different antigen on a target cell, are required to interact together at the cell surface in order to form an active complex which directs demibody-mediated cellular or complement mediated cytotoxicity and/or reporter function and/or therapeutic activity. The demibodies of the present invention are useful in the targeting of particular cells such as cancer cells including leukemic cells, pathogens including malarial, bacterial and viral agents, and stem cells including embryonic and adult stem cells and pathogen cells. The present invention provides, therefore, methods of treatment, diagnosis and undertaking research and compositions comprising demibodies useful for same.
2. Description of the Prior Art
Bibliographic details of the publications referred to by author in this specification are collected at the end of the description.
Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.
A key feature in the search for and development of therapeutic agents is target discrimination or selective toxicity. In particular, the ability to distinguish target cells such as cancer cells or cells infected with pathogen cells amongst a population of normal cells in a subject is of paramount importance. This is particularly the case in cancer therapies where the target cancer cells have many physiological, anatomical and biochemical properties in common with surrounding normal cells. Whilst some anti-cancer drugs do cause collateral damage to normal cells, their use may be indicated or at least justified for particularly aggressive, fast growing cancers.
Therapeutic antibodies are the most rapidly growing area of pharmaceuticals, with more than 30 antibodies in late-phase clinical trials (Hudson and Souriau, Nat Med 9:129-134, 2003). There are many variations of engineered antibodies (e.g. mouse monoclonal, chimeric, humanized, human monoclonal, single chain variable antibody fragments (scFv), minibodies, aptamers). Diabodies developed using recombinant DNA technology, contain two or more single chain variable antibody fragments (scFv) with different binding specificities and appropriate spacing between these domains to enable both scFv to bind antigens concurrently (Hudson and Souriau, supra 2003). Bivalent and bispecific scFv antibodies have been formed using leucine zipper-based dimerization cassettes attached to different scFv (de Kruif and Logtenberg, J Biol Chem 271:7630-7634, 1996). Bi-specific antibodies with different scFv domains connected by a polypeptide chain have been designed to cross-link T-cells with tumours. The two or more interactions that such chimeric antibodies have with different surface antigens on a cell would greatly increase the strength of binding since the dissociation constants for the individual interactions are multiplicative.
Whilst therapeutic antibodies are, important, multi-specific antibodies have not been as successful. There is a need, therefore, to develop antibody-based drugs and other therapeutic agents which are more highly selective for target cells.
Whole antibodies have been proposed as highly specific targeting agents (Carter, Nature Reviews 1:118-128, 2001). In one proposal, cytotoxic agents are linked to an antibody specific for an antigen on a target cell. However, although antibodies have a high degree of target specificity, they have not achieved wide pathological therapeutic use and are primarily used in clinical imaging applications. This may be due to their relatively long circulating half-lives and their associated effector functions.
Modified antibodies, however, have achieved some level of acceptance in immuno-therapeutic applications (Carter (2001) supra; de Haard et al, Adv. Drug Delivery Rev. 31.5-31, 1998; Chames and Baty, FEMS Microbiol. Lett. 189:1-8, 2000; Funaro et al, Biotechnol. Adv. 18:385-401, 2000; Hudson, Exp. Opin. Invest Drugs 9.1231-1242, 2000).
Examples involving therapeutic antibodies are reviewed in Table 1 of Carter (2001) supra.
These antibodies all contain the Fc domain which is required for complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC).
Another useful development in the use of antibody fragments is their fusion to active agents such as radioactive isotypes (Wu et al, Immunotechnology 2:21-36, 1996; Wu et al, Proc. Natl. Acad. Sci. USA 97:8495-8500, 2000; Adams et al, Nuc. Med. Biol. 27:330-346, 2000), enzymes for producing therapy (Bagshawe and Begent, Adv. Drug Delivery Rev. 22:365-367, 1996) and toxins for targeted cell killing (Reiter and Pastan, Tibtech 16:51-520, 1998; Kreitman, Curr. Opin. Immunol. 11:570-578, 1999).
Modified antibodies of particular interest are single chain variable fragments (scFv) carrying the variable region sequences of the light and heavy chains linked together. scFv antibody fragments are derived from Fragment antigen binding (Fab) portions of an antibody comprising the V region of a heavy chain linked by a stretch of synthetic peptide to a V region of a light chain.
Whilst scFv antibody fragments have achieved a reasonable level of utility as targeting molecules, they lack the Fc domain and are unable to induce ADCC or CDC.
The present invention enables modified forms of scFv antibody fragments to be used in targeted cell therapy and/or diagnosis and/or research.